Measurement of pH of solutions, cells and tissues is important in many areas of biological and chemical research and a variety of electrical and spectroscopic techniques have been developed to make such measurements. Measurement of pH by optical indicators, using absorption indicator dyes such as phenolphthalein, has been used routinely in these fields for many years with measurements being made visually or by instrumentation. However, measurement of pH by fluorescence rather than absorbance has the advantage of greater sensitivity, as emitted light is more easily detected than absorbed light. Using fluorescent dyes as pH indicators, it is possible to measure the intracellular pH of single cells by flow cytometry using minimal amounts of the dye.
Many fluorescent dyes useful for measurement of pH are known in the art. The main consideration for a useful fluorescent pH indicator is that the fluorescence intensity of the compound be correlated as reliably as possible with the pH of the medium being measured. An extensive list of readily available fluorescent pH indicators covering the pH range 0 to 14 has been published by G. G. Guilbault in "Practical Fluorescence" (1973).
Many of the fluorescent pH indicators known in the art are phenolic derivatives that undergo absorption shifts to longer wavelength in basic solution, usually with an accompanying increase in fluorescence intensity. Since these rely on deprotonation of a phenol-type functionality to enhance fluorescence intensity, they exhibit a decrease in fluorescence intensity with increasing acidity at longer emission wavelengths. Fluorescein and the umbelliferones are examples of this type of indicator (R. P. Haugland. 1989. "Molecular Probes Handbook" from Molecular Probes, Inc. , Eugene, Oreg., pg. 30, FIGS. 4.2 and 4.3). Few fluorescent pH indicators are known which exhibit increasing fluorescence with increasing acidity, a situation which limits the utility of fluorescent pH indicators in relatively acidic environments such as endosomes and lysosomes.
Seminaphthorhodafluor (SNARF) and seminaphthofluorescein (SNAFL) pH indicators have recently been developed which are useful for measuring pH changes in the range of about 6.3 to 8.6 and are suitable for measurement of intracellular pH. They are benzo[c]xanthene derivatives and are somewhat longer wavelength indicators compared to most other fluorescent pH indicators, making them suitable for use in flow cytometry with excitation by an argon laser at 488 or 514 nm. However, with the exception of SNARF-6 and SNAFL-1, this series of indicators displays the conventional response to pH in which emission intensity decreases with increasing acidity. SNARF-6 and SNAFL-1 exhibit an increase in fluorescence intensity with increasing acidity only at shorter wavelengths, thus limiting detection to more complex and expensive instrumentation. These pH indicators are described in U.S. Pat. No. 4,945,171 and "Molecular Probes Handbook" (R. P. Haugland, 1989, supra, pg. 86-88 and 93; compounds C-1277 and C-1255, respectively). In contrast, the dyes of the present invention exhibit emission spectra in which fluorescence increases with decreasing pH, but this occurs at a longer wavelength emission maximum (i.e., to the right of the isosbestic point). While not wishing to be bound by any particular theory of how the invention operates, Applicants believe the foregoing characteristics suggest that the inventive compounds have a different chemical mechanism for pH dependent fluorescence than SNARF-6 and SNAFL-1.
2',7'-bis-(2-carboxyethyl)-5-(and-6) carboxyfluorescein, (BCECF) is a pH sensitive dye with a side group linked to the fluorescein moiety which consists of two one-carbon spacers attached to carboxylic acids. R. P. Haugland, 1989, supra, pg. 88 and 93, compound B-1151. The side group is similar to that of the present compounds, however, the fluorescein moiety is significantly different structurally from the rhodamine and sulforhodamine moiety of the compounds of the present invention. Further, because BCECF is a derivative of fluorescein, its pH dependent fluorescence intensity is characteristic of fluorescein, i.e., emission intensity decreases with increasing acidity (Graber, et al. 1986. Anal. Biochem. 156, 202-212). BCECF is particularly useful for indicating intracellular pH because it can be synthesized as a BCECF acetoxymethyl ester/monoacetate (BCECF-AM) which is more easily taken up and retained by cells than is BCECF itself (Kolber, et al. 1988. J. Immunol. Mtds. 108, 255-264). Inside the cell BCECF-AM is hydrolyzed by cellular esterases to the acid form which exhibits a pH dependent fluorescence response.
Rhodamine and sulforhodamine type fluorescent dyes are also known in the art and include, for example, rhodamine B, sulforhodamine B, rhodamine 6G, sulforhodamine G, and Texas Red. These dyes are extensively used for fluorescence studies, but they exhibit little, if any, pH dependent fluorescence intensity response. As the inventive compounds are derivatives of rhodamine or sulforhodamine, the pH sensitivity of these compounds was unexpected due to the fact that the parent compounds show pH independent fluorescence. The compounds of the present invention are therefore the first known pH sensitive dyes of the sulforhodamine class. With the possible exception of the SNARF class of dyes, which are hybrids between fluorescein (phenolic) and rhodamine (analine-like) dyes, no rhodamine-based pH sensitive dyes other than those of the present invention are known. In addition, the inventive compounds for the first time provide pH indicators which increase fluorescence intensity with increasing acidity at longer emission wavelengths.